A study was carried out to investigate the effects of different diets for heifers, low- and high-yielding cows on the microbial composition of their faeces and subsequently the impacts of these faeces on CO2 and N2O emissions, N mineralisation and plant N uptake. A diet low in N and high in acid detergent fibre offered to heifers resulted in faeces dominated by fungi. These faeces were characterised by a low content in microbial biomass C and N and a high ergosterol concentration in comparison to the faeces of high-yielding cows. Added to soil, faeces of heifers led to lower emission and stronger N immobilisation during a 14-day incubation in comparison to the faeces of high-yielding cows. Total N2O emission was significantly (P < 0.05) correlated with faecal microbial biomass N. Rye grass yield and N uptake were lowest in the soil supplemented with faeces from heifers in a 62-day pot experiment. Plant N uptake was influenced by the faecal microbial biomass C/N ratio and the fungal C to bacterial C ratio. In conclusion, the faecal microbial biomass was affected to a high degree by the feeding regime and faecal microbial characteristics revealed higher impacts on plant N uptake than soil microbial properties.